RIP3 overexpression sensitizes human breast cancer cells to parthenolide in vitro via intracellular ROS accumulation

Aim： Receptor-interacting protein 3 （RIP3） is involved in tumor necrosis factor receptor signaling, and results in NF-KB-mediated prosurvival signaling and programmed cell death. The aim of this study was to determine whether overexpression of the RIP3 gene could sensitize human breast cancer cells to parthenolide in vitro. Methods： The expression of RIP3 mRNA in human breast cancer cell lines （MCF-7, MDA-MB-231, MDA-MB-435 and T47D） was detected using RT-PCR. Both MDA-MB-231 and MCF-7 cells were transfected with RIP3 expression or blank vectors via lentivirus. Cell viability was measured with MTT assay; intracellular ROS level and cell apoptosis were analyzed using flow cytometry. Results： RIP3 mRNA expression was not detected in the four human breast cancer cell lines tested. However, the transfection induced higher levels of RIP3 protein in MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of RIP3 decreased the IC50 values of parthenolide from 17.6 to 12.6 μmol/L in MCF-7 cells, and from 16.6 to 9.9 μmol/L in MDA-MB-231 cells. Moreover, overexpression of RIP3 significantly increased parthenolide-induced apoptosis and ROS accumulation in MCF-7 and MDA-MB-231 cells. Pretreatment with N-acetyl-cysteine abrogated the increased sensitivity of RIP3-transfected MCF-7 and MDA-MB-231 cells to parthenolide. Conclusion： Overexpression of RIP3 sensitizes MCF-7 and MDA-MB-231 breast cancer cells to parthenolide in vitro via intracellular ROS accumulation.     

Adenovirus-mediated expression of Tob1 sensitizes breast cancer cells to ionizing radiation

Aim： To investigate the effect of the Tob1 gene, a member of the Transducing Molecule of ErbB2/B-cell Translocation Ggene （TOB/BTG） family, by using the adenovirus-mediated expression of Tobl on radiosensitivity in a human breast cancer cell line MDA-MB-231. Methods： Cell survival was determined by clonogenic assay. Apoptosis was evaluated by DNA fragmentation gel electrophoresis and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Protein expression was analyzed by Western blot assay and DNA repair was measured by a host cell reactivation assay. Results： We demonstrated that pre-irradiation treatment with Ad5-Tob1 significantly increased radiosensitivity, accompanying the increased induction of apoptosis and the repression of DNA damage repair. Furthermore, Ad5-Tob1-mediated radiosensitivity correlates with the upregulation of the pro-apoptotic protein Bax and the downregulation of several DNA double strand break repair proteins, including DNA-dependent protein kinases, Ku70 and Ku80, and X-ray-sensitive complementation group 4. Conclusion： Tob1, as a new radiosensitizer, is a new target in the radiotherapy of breast cancer via increasing apoptosis and suppressing DNA repair.     

Quinalizarin induces cycle arrest and apoptosis via reactive oxygen species-mediated signaling pathways in human melanoma A375 cells

Quinalizarin, a bioactive and highly selective compound, is known to promote apoptosis in colon and lung cancer cells. However, studies evaluating quinalizarin-induced apoptosis in melanoma cells have not been conducted. In the present study, we investigated the underlying mechanisms of antimelanoma activity of quinalizarin in human melanoma A375 cells. The MTT assay and Trypan blue staining were used to evaluate the cell viability. The flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Western blot was used to detect the expression of cell cycle and apoptosis-related proteins, MAPK, and STAT3. The results revealed a significant dose and time dependent effect of quinalizarin on inhibiting proliferation in three kinds of human melanoma cells, and had no significant toxic effects on normal cells. Moreover, quinalizarin triggered G2/M phase cell arrest by modulating the protein expression levels of CDK 1/2, cyclin A, cyclin B, p21 and p27, and induced apoptosis by down-regulating the antiapoptotic protein Bcl-2 and upregulating the proapoptotic protein BAD, leading to the activation of caspase-3 and PARP in the caspase cascade in A375 cells. Quinalizarin treatment led to apoptosis of A375 cells via activation of MAPK and inhibition of STAT3 signaling pathways. In addition, quinalizarin increased the level of ROS, but ROS scavenger NAC inhibited quinalizarin-induced apoptosis by regulating MAPK and STAT3 signaling pathways. In summary, quinalizarin induces cell cycle arrest and apoptosis via ROS-mediated MAPK and STAT3 signaling pathways in human melanoma A375 cells, and quinalizarin may be used as a novel and effective antimelanoma therapeutic.     

Suppression of reactive oxygen species‐mediated ERK and JNK activation sensitizes dihydromyricetin‐induced mitochondrial apoptosis in human non‐small cell lung cancer

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer with a high mortality rate and still remains a therapeutic challenge. A strategy for targeting NSCLC is to identify agents that are effective against NSCLC cells while sparing normal cells. Dihydromyricetin (DHM) is the major flavonoid component derived from Ampelopsis grossedentata, which has a long history of use in medicine. Herein, the molecular mechanisms by which DHM exerts its anticancer effects against NSCLC cells were investigated. Results from MTS, colony formation, Western blot, flow cytometric, and JC‐1 mitochondrial membrane potential assays revealed that DHM showed a selective cytotoxic effect against NSCLC cells (A549 and H1975), but not against normal lung (WI‐38) fibroblasts, by inducing apoptosis. DHM‐induced cell apoptosis occurred through Bcl‐w suppression‐mediated mitochondrial membrane depolarization, caspase‐9/‐7/‐3 activation, and poly(ADP‐ribose) polymerase (PARP) cleavage in A549 and H1975 cells. Moreover, treatment of A549 and H1975 cells with DHM induced increase of intracellular peroxide and sustained activation of extracellular signal‐regulated kinase (ERK)1/2 and c‐Jun N‐terminal kinase (JNK)1/2, and the reactive oxygen species scavenger, N‐acetylcysteine (NAC), reversed DHM‐induced ERK and JNK activation. Furthermore, treatment of cells with specific inhibitors of ERK and JNK or NAC significantly promoted the DHM‐induced activation of caspase‐9/‐7/‐3 and PARP cleavage and also sensitized the antitumorigenic effect of DHM on NSCLC cells. These findings define and support a novel function of DHM of inducing mitochondrion‐derived apoptosis in human NSCLC cells, and a combination of DHM with ERK and JNK inhibitors should be a good strategy for preventing NSCLC proliferation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1426–1438, 2017.     

D-氨基葡萄糖衍生物对Eca-109细胞内活性氧、线粒体膜电位的影响

目的：探讨2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖（2-[（3-carboxy-1-oxoprogy1）amino]-2-deoxy-D-Glucose,COPADG）诱导Eca-109细胞凋亡的机制。方法：不同浓度COPADG作用于人食管癌Eca-109细胞24h,检测Eca-109细胞的抑制率、凋亡率、细胞内活性氧（ROS）、线粒体跨膜电位。结果：Eca-109细胞凋亡率与COPADG浓度呈正相关（rs=1.0,P〈0.01）;Eca-109细胞线粒体膜电位水平与Eca-109细胞凋亡率相关（rs=1.0,P〈0.01）;ROS水平与Eca-109细胞凋亡率呈正相关（rs=1.0,P〈0.01）;ROS水平与Eca-109细胞线粒体膜电位水平呈负相关（rs=1.0,P〈0.01）。结论：COPADG可促进Eca-109细胞凋亡,提高Eca-109细胞内ROS水平,并降低线粒体膜电位水平。实验结果提示COPADG提高ROS水平,降低Eca-109细胞线粒体膜电位水平,启动细胞凋亡通路,促使Eca-109细胞凋亡,并且线粒体膜电位的下降是通过提高ROS实现的。     

Growth inhibition, G1-arrest, and apoptosis in MCF-7 human breast cancer cells by novel highly lipophilic 5-fluorouracil derivatives

In this study we evaluate the antitumour activity, the cell cycle arrest and apoptotic properties of novel lipophilic benzene-fused seven-membered 5-fluorouracil (5-FU) analogs in comparison to 5-FU on MCF-7 human breast cancer cells. The lipophilicities of ESB-786B, ESB-252A and ESB-928A were predicted by using the CDR option of the PALLAS 2.0 program. Cytotoxic assays were evaluated in MCF-7 cells treated with the sulforhodamine B colorimetric method. Cell cycle perturbations were studied by flow cytometry. Apoptosis was determined by both DNA fragmentation and annexin V-FITC and propidium iodide staining. The novel derivatives were more lipophilic than 5-FU and induced a marked growth inhibition, in a dose-dependent manner. After treatment with IC(50) value (ranged from 2.5 to 22 microM) for each compound, light microscopy observation showed modifications in the morphology of MCF-7 cells. In addition, the 5-FU analogs arrest cells in the G(0)/G(1) phase of the cell cycle whereas 5-FU induced arrest in S-phase. Moreover, induction of apoptosis was demonstrated by the annexin-V-based assay and confirmed using DNA fragmentation analysis on MCF-7 cells, a cell line in which the induction of DNA laddering is very difficult. The novel benzannelated seven-membered 5-FU analogs can be considered as specific apoptotic inducers. These experimental findings provide support for the use of these novel compounds as new weapons in the fight against breast cancer.     

Silencing of the SNARE protein NAPA sensitizes cancer cells to cisplatin by inducing ERK1/2 signaling, synoviolin ubiquitination and p53 accumulation

We found earlier that NAPA represents an anti-apoptotic protein that promotes resistance to cisplatin in cancer cells by inducing the degradation of the tumor suppressor p53. In the present study, we investigated the cellular mechanism underlying the degradation of p53 by NAPA. Knockdown of NAPA using short-hairpin RNA was shown to induce p53 accumulation and to sensitize HEK293 cells to cisplatin. On the other hand, this sensitization effect was not found in H1299 lung carcinoma cells which lack p53. Expression of exogenous p53 in H1299 cells was increased following knockdown of NAPA and these cells showed increased sensitivity to cisplatin-induced apoptosis. Notably, knockdown of NAPA induced the ubiquitination and degradation of the E3 ubiquitin ligase synoviolin and the accumulation of p53 in unstressed HEK293 cells. Conversely, NAPA overexpression decreased the ubiquitination and degradation of synoviolin, and reduced p53 protein level. Knockdown of NAPA disrupted the interaction between synoviolin and proteins that form the endoplasmic reticulum-associated degradation (ERAD) complex and in turn decreased the ability of this complex to ubiquitinate p53. In addition, knockdown of NAPA induced the activation of the MAPK kinases ERK, JNK and p38, but only inhibition of ERK reduced synoviolin ubiquitination and p53 accumulation. These results indicate that NAPA promotes resistance to cisplatin through synoviolin and the ERAD complex which together induce the degradation of p53 and thus prevent apoptosis. Based on these findings, we propose that the combination of cisplatin and knockdown of NAPA represents a novel and attractive strategy to eradicate p53-sensitive cancer cells.     

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC₅₀ of approximately 80 μM GA at 24 h whereas an IC₅₀ of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 μM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). The percents of MMP (ΔΨ_m) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.     

Codonopsis lanceolata extract induces G0/G1 arrest and apoptosis in human colon tumor HT-29 cells - Involvement of ROS generation and polyamine depletion

Codonopsis lanceolata (Campanulasea) is widely distributed and grown in Asia and has been in use as traditional medicine for long time. The n-butanol fraction (BF) of C. lanceolata significantly inhibited human colon cancer HT-29 cell growth in a dose- and time-dependent manner by inducing G0/G1 phase arrest and apoptosis. The inhibition was associated with intracellular ROS generation and polyamine depletion as evidenced by HPLC quantitatively. Additionally, semi-quantitative RT-PCR revealed enhanced expression of caspase-3, p53, and the Bax/Bcl-2 ratio and reduced expression of survivin in HT-29 cells treated with BF. Furthermore, western blot analysis of p53, JNK, and caspase-3 showed that ROS generation was accompanied by JNK activation. Increase of the Bax/Bcl-2 ratio and activation of caspase-3 might be due to intracellular polyamine depletion. Conclusively, the findings of this study imply a critical role of ROS and polyamine depletion in the anticancer effects of C. lanceolata root extract.     

Ethyl acetate fraction from methanol extraction of Vitis thunbergii var. taiwaniana induced G0/G1 phase arrest via inhibition of cyclins D and E and induction of apoptosis through caspase‐dependent and ‐independent pathways in human prostate carcinoma DU145 cells

Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, belonging to the Vitaceae family and Vitis genus, and widely used as folk herbal medicine. It is traditionally used for the treatment of diarrhea, hypertension, neuroprotection, jaundice, and arthritis. We used the wild‐collected VTT and sterilized them to establish the plant tissue culture, and then took the leaves for DNA sequencing to determine its original base. We use methanol to extract VTT in four different solvents: 1‐butanol, n‐hexane, ethyl acetate, and water. These four preliminary extracts were used to treat human prostate cancer DU145 cells in vitro. We use the flow cytometry to check the cell survival situation. Finally, we found the ethyl acetate layer roughing product (referred VTEA) in human prostate cancer apoptotic effects of cell line DU‐145. In the present studies, we use the crude extract of VTT to examine whether or not it can induce apoptosis of DU145 cells in vitro. Viability assays for extracts of VTT treatment showed that it had dose‐dependent effect on human prostate cancer DU145 cells. We also found that the extract of VTT induces time‐dependent mitochondrial and intrinsic‐dependent apoptosis pathways. The in vitro cytotoxic effects were investigated by cell cycle analysis and the determination of apoptotic DNA fragmentation in DU145 cells. The cell cycle analysis showed that extracts of VTT induced a significant increase in the number of cells in G₀/G₁ phase. The extract of VTT induced chromatin changes and apoptosis of DU145 cells also were confirmed by DAPI and PI staining that were measured by fluorescence microscopy and flow cytometry, respectively. Finally, the expression of relevant proteins was analyzed by Western blot analysis. These results promoted us to further evaluate apoptosis associated proteins and elucidate the possible signal pathway in DU‐145 cells after treated with the extract of VTT.     

Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells

In this report, we investigated the role of oxidative stress in Physalis angulata-induced apoptosis of human oral cancer cells. P. angulata-induced apoptosis was characterized by nuclear morphological changes, membrane blebbing and activation of caspase-9. Exposure of HSC-3 cells to P. angulata caused production of reactive oxygen species and up-regulation of oxidative stress markers heme oxygenase-1 (HO-1), superoxide dismutase (SOD), heat shock protein 70 (HSP70) and caspase-4. Down-regulation of HO-1, SOD and HSP70 proteins expression by attenuation of oxidative stress, pretreatment with glutathione or N-acetylcysteine, significantly decreased P. angulata-triggered cell death. The present study also demonstrated that the mitochondria and the endoplasmic reticulum are the targets of P. angulata in HSC-3 cells. Our results revealed that: (1) reactive oxygen species may play a dominant role in this process, (2) P. angulata induces oxidative stress in HSC-3 cells, (3) P. angulata-initiated apoptosis is caused through oxidative stress-dependent induction of heme oxygenase-1, Cu/Zn SOD and HSP70 proteins expression and (4) antioxidants inhibited P. angulata-induced cell death through inhibition of the proteins expression of HO-1, Cu/Zn SOD and HSP70.     

Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction.     

Implication of NAG-1 in synergistic induction of apoptosis by combined treatment of sodium salicylate and PI3K/MEK1/2 inhibitors in A549 human lung adenocarcinoma cells

Aspirin is used as chemopreventive agents in a variety of human cancer cells including those of colon, lung, breast, and leukemia. Sodium salicylate (NaSal, the natural deacetylated form of aspirin) induced cell cycle arrest and apoptosis in a dose-dependent manner in A549 cells; high dose (20mM) of NaSal-induced apoptosis, whereas low dose (2-10mM) induced cell cycle arrest. We found that NaSal-activated Akt/PKB, ERK1/2, and p38MAPK signal cascades. Twenty micromolar of NaSal-induced apoptotic response of A549 cells was enhanced by the PI3K inhibitors (LY294002 and wortmannin) and in a less extent by the MEK1/2 inhibitors (U0126 and PD98059), whereas it was suppressed by the p38MAPK inhibitor (SB203580). Furthermore, simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could lower the dose of NaSal to induce apoptosis to 2mM in A549 lung cancer cells. Similar enhancement was observed in cells treated with 2mM NaSal and 100muM genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrated that NAG-1 plays a key role in apoptosis by NaSal-based combined treatment. Collectively, our findings indicate that inhibition of the pro-survival Akt/PKB and ERK1/2 signaling may increase the chemopreventive effects of NaSal and combined treatment of two natural compounds (NaSal and genistein) results in a highly synergistic induction of apoptosis, thereby increasing the chemopreventive effects of NaSal against cancer.     

c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH₂-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIP_L/S and Bcl₂ proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl₂ which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl₂ at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death.